Preventives and remedies for pulmonary hypertension

ABSTRACT

The present invention provides a prophylactic and/or therapeutic agent for pulmonary hypertension, comprising an antagonistic mutein of MCP-1 or a salt thereof, a DNA molecule comprising a nucleotide sequence encoding the antagonistic mutein of MCP-1, or a neutralizing antibody against MCP-1. The antagonistic mutein of MCP-1 or a salt thereof, the DNA molecule having a nucleotide sequence encoding the antagonistic mutein of MCP-1, or the neutralizing antibody against MCP-1 has hypotensive activity, and thus is useful as a pharmaceutical agent for preventing and/or treating pulmonary hypertension (primary pulmonary hypertension, in particular).

FIELD OF THE INVENTION

The present invention relates to novel prophylactic and/or therapeuticagents for pulmonary hypertension.

BACKGROUND ART

MPC-1 (Monocyte chemoattractant protein-1: macrophage chemotacticfactor) is a member of the C—C chemokine family and is known to behighly expressed in the arteriosclerotic (e.g. atherosclerotic) lesion(Takeya, M. et al., Hum. Pathol. 24: 534-539 (1993); Yla-Herttuala, S.et al., Proc. Natl. Acad. Sci. USA, 88: 5252-5257 (1991)).

On the other hand, primary pulmonary hypertension (PH) is a disease withpoor prognosis, and heart-lung transplantation is the only therapy forthis disease at present. However, heart-lung transplantation hasconsiderable difficulty being used as a practical treatment due to thelimited supply of donors.

DISCLOSURE OF THE INVENTION

In the view of the situation described above, development of atherapeutic agent that enables treatment of pulmonary hypertension(particularly, primary pulmonary hypertension) without performingheart-lung transplantation has been expected.

MPC-1 (Monocyte chemoattractant protein-1: macrophage chemotacticfactor) is a member of the C—C chemokine family and is known to behighly expressed in the arteriosclerotic (e.g. atherosclerotic) lesion(Takeya, M. et al., Hum. Pathol. 24: 534-539 (1993); Yla-Herttuala, S.et al., Proc. Natl. Acad. Sci. USA, 88: 5252-5257 (1991)).

On the other hand, in the course of primary pulmonary hypertension,inflammation of pulmonary arterioles (inflammatory response mainlyinvolving monocytes/macrophages) develops, and then increase ofpulmonary vascular resistance due to hypertrophy of tunica media, andalso right ventricle hypertrophy follow. The present inventorsdiscovered that an antagonistic mutein of MCP-1 (“antagonistic mutein”may be used herein as a synonym of “dominant negative mutein or mutant”)and the like, which have an inhibitory effect on MCP-1 function asdescribed above, are unexpectedly useful as a prophylactic ortherapeutic agent for primary pulmonary hypertension. They furtherpursued this study and have completed the invention.

The present invention relates to:

(1) A prophylactic and/or therapeutic agent for pulmonary hypertension,comprising an antagonistic mutein of MCP-1 or a salt thereof, a DNAmolecule having a nucleotide sequence encoding the antagonistic muteinof MCP-1, or a neutralizing antibody against MCP-1;

(2) The agent described in (1), wherein said antagonistic mutein ofMCP-1 is 7ND-MCP-1;

(3) The agent described in (1), wherein said pulmonary hypertension isprimary pulmonary hypertension;

(4) Use of the antagonistic mutein of MCP-1 or a salt thereof, a DNAmolecule having a nucleotide sequence encoding the antagonistic muteinof MCP-1, or a neutralizing antibody against MCP-1 described in (1) forproducing a prophylactic and/or therapeutic agent for pulmonaryhypertension; and

(5) A method for preventing and/or treating pulmonary hypertension,comprising administration of the antagonistic mutein of MCP-1 or a saltthereof, or a nucleotide sequence encoding the antagonistic mutein ofMCP-1 described in (1) to mammals.

The antagonistic mutein of MCP-1 of the present invention (hereinaftermay be referred only to as “the muteins of the invention”) may be anymutein that has an inhibitory effect on the function (e.g.monocyte/macrophage chemotactic (migration) function) of MCP-1 (Rollins,B. J., Chemokines. Blood. 90: 909-928 (1997); a protein consisting ofthe amino acid sequence set forth in SEQ ID NO: 1). Specifically, theyinclude:

(1) A mutein consisting of the partial amino acid sequence correspondingto the residues 2-76 from the N-terminus of MCP-1;

(2) A mutein consisting of the partial amino acid sequence correspondingto the residues 3-76 from the N-terminus of MCP-1;

(3) A mutein consisting of the partial amino acid sequence correspondingto the residues 4-76 from the N-terminus of MCP-1;

(4) A mutein consisting of the partial amino acid sequence correspondingto the residues 5-76 from the N-terminus f MCP-1;

(5) A mutein consisting of the partial amino acid sequence correspondingto the residues 6-76 from the N-terminus of MCP-1;

(6) A mutein consisting of the partial amino acid sequence correspondingto the residues 7-76 from the N-terminus of MCP-1;

(7) A mutein consisting of the partial amino acid sequence correspondingto the residues 8-76 from the N-terminus of MCP-1;

(8) A mutein consisting of the partial amino acid sequence correspondingto the residues 9-76 from the N-terminus of MCP-1;

(9) A mutein consisting of the partial amino acid sequence correspondingto the residues 10-76 from the N-terminus of MCP-1;

(10) A mutein consisting of the partial amino acid sequencecorresponding to the residues 11-76 from the N-terminus of MCP-1;

(11) A mutein consisting of the amino acid sequence in which theresidues 2-8 from the N-terminus of MCP-1 are deleted (SEQ ID NO: 2)(hereinafter referred to as “7ND-MCP-1”);

(12) A mutein in which Asp, the third residue from the N-terminus ofMCP-1, is replaced with Ala;

(13) A mutein in which Val, the 22nd residue from the N-terminus ofMCP-1, is replaced with Asp; and

(14) A mutein in which Arg, the 24th residue from the N-terminus ofMCP-1, is replaced with Leu.

In particular, 7ND-MCP-1 is preferabley used.

The mutein of the present invention may be a mutein of protein derivedfrom any cells (e.g., retina cells, liver cells, splenocytes, nervecells, glial cells, β cells of pancreas, bone marrow cells, mesangialcells, Langerhans' cells, epidermic cells, epithelial cells, endothelialcells, fibroblasts, fibrocytes, myocytes, fat cells, immune cells (e.g.,macrophage, T cells, B cells, natural killer cells, mast cells,neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovialcells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary glandcells, hepatocytes or interstitial cells, the corresponding precursorcells, stem cells, cancer cells, etc.), or any tissues where such cellsare present, e.g., brain or any region of the brain (e.g., olfactorybulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus,hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata,cerebellum), spinal cord, hypophysis, stomach, pancreas, kidney, liver,gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle,lung, gastrointestinal tract (e.g., large intestine, small intestine,intestine duodenum), blood vessel, heart, thymus, spleen, submandibulargland, peripheral blood, peripheral blood cells, prostate, testis,ovary, placenta, uterus, bone, joint, skeletal muscle, etc. from humanand other mammalians (e.g., guinea pigs, rats, mice, rabbits, swine,sheep, bovine, monkeys, etc.). The mutein may also be a syntheticprotein.

The mutein of the present invention may have substantially the sameamino acid sequence as that shown by SEQ ID NO: 2 as long as it caninhibit the activity of MCP-1 (for example, macrophage chemotacticactivity). The substantially same amino acid sequence as that shown bySEQ ID NO: 2 includes, for example, amino acid sequences having about40% or more, preferably about 60% or more, more preferably about 80% ormore, further preferably about 90% or more, most preferably about 95% ormore homology with the amino acid sequence shown by SEQ ID NO: 2.

The mutein of the present invention also includes a mutein comprising(i) an amino acid sequence having deletion of 1 or more (preferably 1 toabout 20, more preferably 1 to about 9, further preferably several (1 to5)) amino acids in the amino acid sequence shown by SEQ ID NO: 2, (ii)an amino acid sequence having addition of 1 or more (preferably 1 toabout 20, more preferably 1 to about 9, further preferably several (1 to5)) amino acids in the amino acid sequence shown by SEQ ID NO: 2, (iii)an amino acid sequence having substitution of 1 or more (preferably 1 toabout 20, more preferably 1 to about 9, further preferably several (1 to5)) amino acids with other amino acids in the amino acid sequence shownby SEQ ID NO: 2, or (iv) an amino acid sequence having a combination ofthe above modifications. When the amino acid sequence is modified viaaddition or deletion of amino acid residues, positions for the additionor deletion are not specifically limited.

The muteins of the present invention are represented in accordance withthe conventional way of describing peptides, with the N-terminus (aminoterminus) on the left side and the C-terminus (carboxyl terminus) on theright side. In the muteins of the present invention, including themutein consisting of the amino acid sequence shown by SEQ ID NO:2, theC-terminus is usually in the form of a carboxyl group (—COOH) or acarboxylate (—COO⁻) but may be in the form of an amide (—CONH₂) or anester (—COOR).

Examples of the ester group shown by R include a C₁₋₆alkyl group such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C₃₋₈cycloalkylgroup such as cyclopentyl, cyclohexyl, etc.; a C₆₋₁₂ aryl group such asphenyl, α-naphthyl, etc.; a C₇₋₁₄ aralkyl group such as aphenyl-C₁₋₂-alkyl group, e.g., benzyl, phenethyl, etc., or anα-naphthyl-C₁₋₂-alkyl group such as α-naphthylmethyl, etc.; and thelike. In addition, pivaloyloxymethyl or the like, which is used widelyas an ester for oral administration, may also be used.

When the mutein of the present invention contains a carboxyl group (or acarboxylate) at a position other than the C-terminus, it may be amidatedor esterified and such an amide or ester is also included within themutein of the present invention. The ester group may be the same groupas that described with respect to the C-terminus described above.

Furthermore, the muteins of the present invention include variants ofthe above-mentioned muteins, wherein the amino group at the N-terminalmethionine residue of the mutein supra is protected with a protectinggroup (for example, a C₁₋₆ acyl group such as a C₂₋₆ alkanoyl group,e.g., formyl group, acetyl group, etc.); those wherein the N-terminalregion is cleaved in vivo and the glutamyl group thus formed ispyroglutaminated; those wherein a substituent (e.g., —OH, —SH, aminogroup, imidazole group, indole group, guanidino group, etc.) on the sidechain of an amino acid in the molecule is protected with a suitableprotecting group (e.g., a C₁₋₆ acyl group such as a C₂₋₆ alkanoyl group,e.g., formyl group, acetyl group, etc.), or conjugated proteins such asglycoproteins bound to sugar chains.

A salt of the mutein of the present invention is a physiologicallyacceptable salt with an acid or base. Especially, a physiologicallyacceptable acid addition salt is preferred. Examples of the saltinclude, for example, a salt with an inorganic acid (e.g., hydrochloricacid, phosphoric acid, hydrobromic acid, sulfuric acid) or with anorganic acid (e.g., acetic acid, formic acid, propionic acid, furmaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonicacid).

The mutein of the present invention or a salt thereof may be produced bypreparing MCP-1 via a known purification method from human or othermammalian cells or tissues as described above, and then deleting anamino acid residue or a partial amino acid sequence from MCP-1 inaccordance with a known method; or by culturing a transformant having aDNA encoding the mutein of the present invention, as later described.Furthermore, the mutein or its salt may also be produced by a method forprotein synthesis as described below or a modified method thereof.

When MCP-1 is produced from human or mammalian tissues or cells, afterhomogenization of human or mammalian tissues or cells, extraction withan acid or the like, and isolation and purification by a combination ofchromatography techniques such as reverse phase chromatography, ionexchange chromatography, and the like are carried out.

To synthesize the mutein of the present invention, commerciallyavailable resins that are used for protein synthesis may be used.Examples of such resins include chloromethyl resin, hydroxymethyl resin,benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcoholresin, 4-methylbenzhydrylamine resin, PAM resin,4-hydroxymethylmehtylphenyl acetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Usingthese resins, amino acids in which α-amino groups and functional groupson the side chains are appropriately protected are condensed on theresin in the order of the sequence of the objective mutein according tovarious condensation methods publicly known in the art. At the end ofthe reaction, the mutein is cut out from the resin and at the same time,the protecting groups are removed. Then, intramolecular disulfidebond-forming reaction is performed in a highly diluted solution toobtain the objective mutein or its amides.

For condensation of the protected amino acids described above, a varietyof activation reagents for protein synthesis may be used, andcarbodiimides are particularly preferable. Examples of suchcarbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids in combination with aracemization inhibitor (e.g., HOBt, HOOBt) are added directly to theresin, or the protected amino acids are previously activated in the formof symmetric acid anhydrides, HOBt esters or HOOBt esters, followed byadding the thus activated protected amino acids to the resin.

Solvents suitable for use to activate the protected amino acids orcondense with the resin may be chosen from solvents known to be usablefor protein condensation reactions. Examples of such solvents are acidamides such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylenechloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.;sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine,dioxane, tetrahydrofuran, etc.; nitrites such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to proteinbinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. The condensation is examinedby a test using the ninhydrin reaction; when the condensation isinsufficient, the condensation can be completed by repeating thecondensation reaction without removal of the protecting groups. When thecondensation is yet insufficient even after repeating the reaction,unreacted amino acids are acetylated with acetic anhydride oracetylimidazole to avoid adverse effect on the subsequent reaction.

Examples of the protecting groups used to protect the amino groups ofthe starting compounds include Z, Boc, t-pentyloxycarbonyl,isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z,adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc.

A carboxyl group can be protected by, e.g., alkyl esterification (in theform of linear, branched or cyclic alkyl esters of the alkyl moiety suchas methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, etc.), aralkyl esterification(e.g., esterification in the form of benzyl ester, 4-nitrobenzyl ester,4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, etc.),phenacyl esterification, benzyloxycarbonyl hydrazidation,t-butoxycarbonyl hydrazidation, trityl hydrazidation, or the like.

The hydroxyl group of serine can be protected through, for example, itsesterification or etherification. Examples of groups appropriately usedfor the esterification include a lower alkanoyl group, such as acetylgroup, an aroyl group such as benzoyl group, and a group derived fromcarbonic acid such as benzyloxycarbonyl group, ethoxycarbonyl group,etc. Examples of a group appropriately used for the etherificationinclude benzyl group, tetrahydropyranyl group, t-butyl group, etc.

Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting compoundsinclude the corresponding acid anhydrides, azides, activated esters(esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)). As the activated aminoacids, in which the amino groups are activated in the starting material,the corresponding phosphoric amides are employed.

To eliminate (remove) the protecting groups, there are used catalyticreduction under hydrogen gas flow in the presence of a catalyst such asPd-black or Pd-carbon; an acid treatment with anhydrous hydrogenfluoride, methanesulfonic acid, trifluoromethane-sulfonic acid ortrifluoroacetic acid, or a mixture solution of these acids; a treatmentwith a base such as diisopropylethylamine, triethylamine, piperidine orpiperazine; and reduction with sodium in liquid ammonia. The eliminationof the protecting group by the acid treatment described above is carriedout generally at a temperature of approximately −20° C. to 40° C. In theacid treatment, it is efficient to add a cation scavenger such asanisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide,1,4-butanedithiol or 1,2-ethanedithiol. Furthermore, 2,4-dinitrophenylgroup known as the protecting group for the imidazole of histidine isremoved by a treatment with thiophenol. Formyl group used as theprotecting group of the indole of tryptophan is eliminated by theaforesaid acid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol, as well as by a treatment with an alkali such as adilute sodium hydroxide solution and dilute ammonia.

Protection of functional groups that should not be involved in thereaction of the starting materials, protecting groups, elimination ofthe protecting groups and activation of functional groups involved inthe reaction may be appropriately selected from known groups and knownmeans.

In another method for obtaining an amide of the mutein, for example, theα-carboxyl group of the carboxy terminal amino acid is first protectedby amidation; the peptide (protein) chain is then extended from theamino group side to a desired length. Thereafter, a protein in whichonly the protecting group of the N-terminal α-amino group in the peptidechain has been eliminated from the protein and a protein in which onlythe protecting group of the C-terminal carboxyl group has beeneliminated are prepared. The two proteins are condensed in a mixture ofthe solvents described above. The details of the condensation reactionare the same as described above. After the protected protein obtained bythe condensation is purified, all the protecting groups are eliminatedby the method described above to give the desired crude protein. Thiscrude protein is purified by various known purification means.Lyophilization of the major fraction gives an amide of the desiredmutein.

To prepare an ester of the mutein, for example, the α-carboxyl group ofthe carboxy terminal amino acid is condensed with a desired alcohol toprepare the amino acid ester, which is followed by procedure similar tothe preparation of the amidated mutein above to give an ester form ofthe desired mutein.

The mutein of the present invention can be produced by a known methodfor peptide synthesis, or by cleaving MCP-1 with an appropriatepeptidase. For the method for peptide synthesis, for example, eithersolid phase synthesis or liquid phase synthesis may be used. That is, apartial peptide or an amino acid that can compose the mutein of thepresent invention is condensed with the remaining part. When the productcontains protecting groups, these protecting groups are removed to givethe desired mutein. Publicly known methods for condensation andelimination of the protecting groups are described in 1)-5) below.

1) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, IntersciencePublishers, New York (1966)

2) Schroeder & Luebke: The Peptide, Academic Press, New York (1965)

3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics andexperiments of peptide synthesis), published by Maruzen Co. (1975)

4) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza(Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry ofProteins) IV, 205 (1977)

5) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu (A sequel toDevelopment of Pharmaceuticals), Vol. 14, Peptide Synthesis, publishedby Hirokawa Shoten

After completion of the reaction, the mutein of the present inventionmay be purified and isolated by a combination of conventionalpurification methods such as solvent extraction, distillation, columnchromatography, liquid chromatography and recrystallization. When themutein obtained by the above method is in a free form, the mutein can beconverted into an appropriate salt by a publicly known method; when themutein is obtained in a salt form, it can be converted into a free formby a publicly known method.

The DNA encoding the mutein of the present invention may be any DNAcontaining a nucleotide sequence encoding the mutein, and may also bederived from any of genomic DNA, genomic DNA library, cDNA derived fromthe cells and tissues described above, cDNA library derived from thecells and tissues described above and synthetic DNA. The vector to beused for the library may be any of bacteriophage, plasmid, cosmid andphagemid. The DNA may also be directly amplified by reversetranscriptase polymerase chain reaction (hereinafter abbreviated asRT-PCR) using the total RNA or mRNA fraction prepared from the cells andtissues described above.

Specifically, the DNA encoding MCP-1 consisting of the amino acidsequence shown by SEQ ID NO: 1 includes a DNA having the nucleotidesequence shown by SEQ ID NO: 3. The DNA encoding 7ND-MCP-1 consisting ofthe amino acid sequence shown by SEQ ID NO: 2 includes a DNA having thenucleotide sequence shown by SEQ ID NO: 4.

For cloning of the DNA that encodes the mutein of the present invention,the DNA may be either amplified by PCR using synthetic DNA primerscontaining a part of the nucleic acid sequence encoding the mutein ofthe present invention, or the DNA inserted into an appropriate vectorcan be selected by hybridization with a labeled DNA fragment or alabeled synthetic DNA that encodes a part or entire region of the muteinof the present invention. The hybridization can be carried out, forexample, according to the method described in Molecular Cloning, 2nd, J.Sambrook et al., Cold Spring Harbor Lab. Press, 1989. The hybridizationmay also be performed using a commercially available library inaccordance with the protocol described in the attached instruction.

Modification (deletion, addition, substitution) of the DNA sequence canbe effected in accordance with a publicly known method such as theGupped duplex method or the Kunkel method or its modification, using apublicly known kit available as Mutan™-G or Mutan™-K (Takara Shuzo Co.,Ltd.).

The cloned DNA encoding the mutein of the present invention can be useddepending upon its purpose, as it is or if desired, after digestion witha restriction enzyme or after addition of a linker thereto. The DNA maycontain ATG as a translation initiation codon at the 5′ end thereof andmay further contain TAA, TGA or TAG as a translation termination codonat the 3′ end thereof. These translation initiation and terminationcodons may also be added by using an appropriate synthetic DNA adapter.

The expression vector for the mutein of the present invention can beproduced, for example, by (a) excising the desired DNA fragment from theDNA encoding the mutein of the present invention, and then (b) ligatingthe DNA fragment downstream of a promoter in an appropriate expressionvector.

Examples of the vector include plasmids derived form E. coli (e.g.,pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis(e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19,pSH15), bacteriophages such as λ-phage, etc., animal viruses such asretrovirus, vaccinia virus, baculovirus, etc. as well as pA1-11, pXT1,pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

The promoter used in the present invention may be any promoter suitablefor a host to be used for gene expression. In the case of using animalcells as the host, examples of the promoter include SRα promoter, SV40promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc. Among them,CMV promoter or SRα promoter is preferably used. When the host isbacteria of the genus Escherichia, preferred examples of the promoterinclude trp promoter, lac promoter, recA promoter, λP_(L) promoter, lpppromoter, etc. In the case of using bacteria of the genus Bacillus asthe host, preferred example of the promoter are SPO1 promoter, SPO2promoter and penP promoter. When yeast is used as the host, preferredexamples of the promoter are PHO5 promoter, PGK promoter, GAP promoterand ADH promoter. When insect cells are used as the host, preferredexamples of the promoter include polyhedrin prompter and P10 promoter.

In addition to the foregoing examples, the expression vector may furtheroptionally contain an enhancer, a splicing signal, a poly A additionsignal, a selection marker, SV40 replication origin (hereinaftersometimes abbreviated as SV40ori) etc. Examples of the selection markerinclude dihydrofolate reductase (hereinafter sometimes abbreviated asdhfr) gene, ampicillin resistant gene (hereinafter sometimes abbreviatedas Amp^(r)), neomycin resistant gene (hereinafter sometimes abbreviatedas Neo^(r), G418 resistance), etc. The dhfr gene confers methotrexate(MTX) resistance and Neo gene confers G418 resistance. In particular,when dhfr gene is used as the selection marker in dhfr⁻ Chinese HamsterOvary (CHO) cells, the target gene can also be selected in athymidine-free medium.

If necessary and desired, a signal sequence that matches with a host isadded to the N-terminus of the mutein of the present invention. Examplesof the signal sequence that can be used are Pho A signal sequence, OmpAsignal sequence, etc. in the case of using bacteria of the genusEscherichia as the host; α-amylase signal sequence, subtilisin signalsequence, etc. in the case of using bacteria of the genus Bacillus asthe host; MFα signal sequence, SUC2 signal sequence, etc. in the case ofusing yeast as the host; and insulin signal sequence, α-interferonsignal sequence, antibody molecule signal sequence, etc. in the case ofusing animal cells as the host, respectively.

Using the vector containing the DNA encoding the mutein of the presentinvention thus constructed, a transformant can be manufactured.

Examples of the host, which may be employed, are bacteria belonging tothe genus Escherichia, bacteria belonging to the genus Bacillus, yeast,insect cells, insects and animal cells, etc.

Specific examples of the bacteria belonging to the genus Escherichiainclude Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221 (Journalof Molecular Biology, 120, 517 (1978)), HB101 (Journal of MolecularBiology, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954)), etc.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 (Journal ofBiochemistry, 95, 87 (1984)), etc.

Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris KM71, etc.

Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cells (Sf cells), MG1 cells derived from mid-intestine ofTrichoplusia ni, High Five™ cells derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cells (BmN cells),etc. are used. Examples of the Sf cell which can be used are Sf9 cells(ATCC CRL1711) and Sf21 cells (both cells are described in Vaughn, J. L.et al., In Vitro, 13 213-217 (1977).

As the insect, for example, a larva of Bombyx mori can be used (Maeda,et al., Nature, 315, 592 (1985)).

Examples of animal cells include monkey cells COS-7, Vero, Chinesehamster ovary cells (hereinafter referred to as CHO cells), dhfrgene-deficient Chinese hamster ovary cells (hereinafter simply referredto as CHO(dhfr⁻) cell), mouse L cells, mouse AtT-20, mouse myelomacells, rat GH3, human FL cells, 293 cells, C127 cells, BALB3T3 cells,Sp-2 cells, etc. Among those, CHO cells, CHO (dhfr⁻) cells, 293 cellsare preferred.

Bacteria belonging to the genus Escherichia can be transformed, forexample, by the method described in Proc. Natl. Acad. Sci. U.S.A., 69,2110 (1972) or Gene, 17, 107 (1982).

Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979).

Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

Insect cells or insects can be transformed, for example, according tothe method described in Bio/Technology, 6, 47-55(1988), etc.

Animal cells can be transformed, for example, according to the methoddescribed in Saibo Kogaku (Cell Engineering), extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267 (1995), published by Shujunsha, or Virology, 52, 456 (1973).

The method of introducing the expression vector into cells includes, forexample, calcium phosphate method (Graham, F. L. and van der Eb, A. J.Virology, 52, 456-467 (1973)), eectroporation (Nuemann, E. et al. EmboJ., 1, 841-845 (1982)),etc.

Thus, a transformant transformed with the expression vector containingthe DNA encoding the mutein of the present invention can be obtained.

Furthermore, to express the mutein of the present invention in a stablemanner using animal cells, the animal cell clone can be selected, thechromosome of which the introduced expression vector is incorporatedinto. To be more specific, using the above selection marker as an index,a transformant can be selected. From these animal cells obtained by useof the selection marker, it is possible to obtain a stable animal cellstrain expressing highly the mutein of the present invention byrepeating the clonal selection. Moreover, when using dhfr gene as aselection marker, the cells are cultured in gradually increasedconcentrations of MTX, and a MTX-resistant cell strain is selected. Inthis way, it is possible to obtain an animal cell strain with higherexpression by amplifying the DNA encoding the mutein as well as dhfrgene in the cell.

The mutein of the present invention can be produced by cultivating theabove-mentioned transformant under condition capable of expressing theDNA encoding the mutein of the present invention; and producing andaccumulating the mutein of the present invention.

When the host is bacteria belonging to the genus Escherichia or thegenus Bacillus,the transformant can be appropriately incubated in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, and so on. Examples of the carbon sources include glucose,dextrin, soluble starch, sucrose, etc. Examples of the nitrogen sourcesinclude inorganic or organic materials such as ammonium salts, nitratesalts, corn steep liquor, peptone, casein, meat extract, soybean cake,potato extract, etc. Examples of the inorganic materials are calciumchloride, sodium dihydrogenphosphate, magnesium chloride, etc. Inaddition, yeast, vitamins, growth promoting factors etc. may also beadded to the medium. Preferably, pH of the medium is adjusted to about 5to about 8.

A preferred example of the medium for incubation of the bacteriabelonging to the genus Escherichia is M9 medium supplemented withglucose and Casamino acids (Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972). Ifnecessary and desired, a chemical such as 3β-indolylacrylic acid can beadded to the medium thereby to activate the promoter efficiently.

When the bacteria belonging to the genus Escherichia are used as thehost, the transformant is usually cultivated at about 15° C. to about43° C. for about 3 hours to about 24 hours. If necessary and desired,the culture may be aerated or agitated.

When the bacteria belonging to the genus Bacillus are used as the host,the transformant is cultivated generally at about 30° C. to about 40° C.for about 6 hours to about 24 hours. If necessary and desired, theculture can be aerated or agitated.

When yeast is used as the host, the transformant is cultivated, forexample, in Burkholder's minimal medium (Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)) or in SD medium supplementedwith 0.5% Casamino acids (Bitter, G. A. et al., Proc. Natl. Acad. Sci.U.S.A., 81, 5330 (1984)). Preferably, pH of the medium is adjusted toabout 5 to about 8. In general, the transformant is cultivated at about20° C. to about 35° C. for about 24 hours to about 72 hours. Ifnecessary and desired, the culture can be aerated or agitated.

When insect cells are used as the host, the transformant is cultivatedin, for example, Grace's Insect Medium (Grace, T. C. C., Nature, 195,788 (1962)) to which an appropriate additive such as immobilized 10%bovine serum is added. Preferably, pH of the medium is adjusted to about6.2 to about 6.4. Normally, the transformant is cultivated at about 27°C. for about 3 days to about 5 days and, if necessary and desired, theculture can be aerated or agitated.

When animal cells are employed as the host, the transformant iscultivated in, for example, MEM medium containing about 5% to about 20%fetal bovine serum (Science, 122, 501 (1952)), DMEM medium (Virology, 8,396 (1959)), RPMI 1640 medium (The Journal of the American MedicalAssociation, 199, 519 (1967)), 199 medium (Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)), etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultivated at about 30° C. to about 40° C. for about 15 hours to about72 hours and, if necessary and desired, the culture can be aerated oragitated.

When using CHO (dhfr⁻) cells and dhfr gene as a selection marker,thymidine-free DMEM medium containing dialyzed fetal bovine serum ispreferred.

The mutein of the present invention can be separated and purified fromthe culture described above by the following procedures.

When the mutein of the present invention is extracted from the cultureof bacteria or cells, after the culture of bacteria or cells arecollected by a publicly known method and suspended in a appropriatebuffer. The bacteria or cells are then disrupted by publicly knownmethods such as ultrasonication, a treatment with lysozyme and/orfreeze-thaw cycling, followed by centrifugation, filtration, etc. Thus,the crude extract of the mutein of the present invention can beobtained. The buffer used for the procedure may contain a proteindenaturing agent such as urea or guanidine hydrochloride, or asurfactant such as Triton X-100™, etc.

When the protein is secreted into the culture medium, after completionof the cultivation, the supernatant can be separated from the bacteriaor cells to collect the supernatant by a publicly known method.

The mutein contained in the supernatant or the extract thus obtained canbe purified by appropriately combining the publicly known methods forseparation and purification. Such publicly known methods for separationand purification include a method utilizing difference in solubilitysuch as salting out, solvent precipitation, etc.; a method utilizingmainly difference in molecular weight such as dialysis, ultrafiltration,gel filtration, SDS-polyacrylamide gel electrophoresis, etc.; a methodutilizing difference in electric charge such as ion exchangechromatography, etc.; a method utilizing difference in specific affinitysuch as affinity chromatography, etc.; a method utilizing difference inhydrophobicity such as reverse phase high performance liquidchromatography, etc.; a method utilizing difference in isoelectric pointsuch as isoelectrofocusing electrophoresis; and the like.

When the mutein thus obtained is in a free form, it can be convertedinto a salt form by a publicly known method or modification thereof. Onthe other hand, when the mutein is obtained in a salt form, it can beconverted into a free form or another salt form by a publicly knownmethod or modification thereof.

The mutein produced by the recombinant can be treated, before or afterthe purification, with an appropriate protein-modifying enzyme so thatthe mutein can be appropriately modified or deprived of a partialpolypeptide. Examples of the protein-modifying enzyme include trypsin,chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase or thelike.

The presence of the thus produced mutein of the present invention can bedetermined by an enzyme immunoassay using a specific antibody, or thelike.

Neutralizing antibodies to MCP-1 (referred to as the antibodies of thepresent invention) may be any of polyclonal antibodies and monoclonalantibodies which are capable of neutralizing MCP-1 and recognizingMCP-1.

The antibodies of the present invention may be manufactured by apublicly known method for manufacturing antibodies or antisera, usingMCP-1 as an antigen.

[Preparation of Monoclonal Antibody]

(a) Preparation of Monoclonal Antibody-producing Cells

MCP-1 is administered to warm-blooded animals either solely or togetherwith carriers or diluents to the site where the production of antibodyis possible by the administration. In order to potentiate the antibodyproductivity upon the administration, complete Freund's adjuvants orincomplete Freund's adjuvants may be administered. The administration isusually carried out once in every 2 to 6 weeks and 2 to 10 times intotal. Examples of the applicable warm-blooded animals are monkeys,rabbits, dogs, guinea pigs, mice, rats, sheep and goats, chicken withmice and rats being preferred.

In the preparation of monoclonal antibody-producing cells, warm-bloodedanimals, e.g., mice are immunized with an antigen, the individual, inwhich the antibody titer is detected, is selected, then the spleen orlymph node is collected after 2 to 5 days from the final immunization.Antibody-producing cells contained therein are fused with myeloma cellsto give monoclonal antibody-producing hybridomas. Measurement of theantibody titer in antisera may be made, for example, by reacting alabeled form of the protein, which will be described later, with theantiserum followed by assaying the binding activity of the labelingagent bound to the antibody. The fusion may be carried out by a knownmethod, for example, by Koehler and Milstein method (Nature, 256 495,1975). Examples of the fusion accelerator are polyethylene glycol (PEG),Sendai virus, etc., of which PEG is preferably employed.

Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1, etc., i.e.ones derived from warm-blooded animals. In particular, P3U1 ispreferably employed. A preferred ratio of the count of theantibody-producing cells used (spleen cells) to the count of myelomacells is within a range of approximately 1:1 to 20:1. PEG (preferably,PEG 1000 to PEG 6000) may be added in a concentration of approximately10 to 80%. An efficient cell fusion can be carried out by incubating at20 to 40° C., preferably at 30 to 37° C. for 1 to 10 minutes.

Various methods can be used for screening of a monoclonalantibody-producing hybridoma. Examples of such methods include a methodwhich comprises adding the supernatant of hybridoma to a solid phase(e.g., microplate) adsorbed with the protein as an antigen directly ortogether with a carrier, adding an anti-immunoglobulin antibody (whenmouse cells are used for the cell fusion, anti-mouse immunoglobulinantibody is used) labeled with a radioactive substance or an enzyme, orProtein A and detecting the monoclonal antibody bound to the solidphase, and a method which comprises adding the supernatant of hybridomato a solid phase adsorbed with an anti-immunoglobulin antibody orProtein A, adding the protein labeled with a radioactive substance or anenzyme and detecting the monoclonal antibody bound to the solid phase.

The monoclonal antibody can be selected by publicly known methods or bymodifications of these methods. In general, the selection can beeffected in a medium for animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any selection and growthmedium can be employed as far as the hybridoma can grow therein. Forexample, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1% to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.) and thelike can be used for the selection and growth medium. The cultivation iscarried out generally at 20° C. to 40° C., preferably at about 37° C.,for 5 days to 3 weeks, preferably 1 to 2 weeks. The cultivation can beconducted normally in 5% CO₂. The antibody titer of the culturesupernatant of hybridomas can be determined as in the assay for theantibody titer in antisera described above.

(b) Purification of Monoclonal Antibody

Separation and purification of a monoclonal antibody can be carried outby a known method, for example, according to a method for separation andpurification of immunoglobulins [e.g., salting-out, alcoholprecipitation, isoelectric point precipitation, electrophoresis,adsorption and desorption with ion exchangers (e.g., DEAE),ultracentrifugation, gel filtration, or a specific purification methodwhich comprises collecting only an antibody with an activated adsorbentsuch as an antigen-binding solid phase, Protein A, Protein G, etc. anddissociating the binding to obtain the antibody].

[Preparation of Polyclonal Antibody]

The polyclonal antibody of the present invention can be manufactured bya publicly known method or modification thereof. For example, a complexof immunogen (a protein antigen) and a carrier protein is prepared, anda warm-blooded animal is immunized with the complex in a manner similarto the method described above for the manufacture of monoclonalantibodies. A material containing the polyclonal antibody is collectedfrom the immunized animal, and then separation and purification of theantibody are performed.

In the complex of an immunogen and a carrier protein used to immunize awarm-blooded animal, the type of carrier protein and the mixing ratio ofa carrier to hapten may be any type and in any ratio, as long as theantibody is efficiently produced to the immunized hapten crosslinked tothe carrier. For example, bovine serum albumin, bovine thyroglobulins,keyhole limpet hemocyanin, etc. is coupled to a hapten in acarrier-to-hapten weight ratio of about 0.1 to 20, preferably about 1 to5.

A variety of condensing agents can be used for the coupling of a carrierto hapten. Glutaraldehyde, carbodiimide, maleimide activated ester,activated ester reagents containing thiol group or dithiopyridyl group,etc. are used for the coupling.

The condensation product is administered to warm-blooded animals eitheralone or together with a carrier or diluent to the site where theantibody-can be produce by the administration. In order to potentiatethe antibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once approximately in every 2 to 6 weeksand about 3 to 10 times in total.

The polyclonal antibody can be collected from the blood, ascites, etc.,preferably from the blood of warm-blooded animals immunized according tothe method described above.

The polyclonal antibody titer in antiserum can be assayed by the sameprocedure as that for the determination of serum antibody titerdescribed above. The separation and purification of the polyclonalantibody can be carried out, following the method for separation andpurification of immunoglobulins as described in the separation andpurification of the monoclonal antibodies.

The mutein of the present invention, the DNA encoding the mutein, or theantibody of the present invention can be used as a prophylactic and/ortherapeutic agent for pulmonary hypertension, more specifically, primarypulmonary hypertension.

When the DNA encoding the mutein of the present invention is used as theprophylactic/therapeutic agent described above, the DNA is administeredby itself; alternatively, the DNA is inserted into an appropriate vectorsuch as retrovirus vector, adenovirus vector, adenovirus-associatedvirus vector, etc. and then administered in a conventional manner. TheDNA of the present invention may also be administered as naked DNA orwith an adjuvant to assist its uptake, by gene gun or through a cathetersuch as a catheter with a hydrogel.

When the mutein of the present invention is used as theprophylactic/therapeutic agent described above, the mutein may be usedat purity level of at least 90%, preferably at least 95%, morepreferably at least 98%, further preferably 99%.

When the mutein of the present invention or the antibody of the presentinvention is used as the prophylactic/therapeutic agent described above,it can be used orally, for example, in the form of tablet which may besugar-coated if necessary and desired, capsule, elixir, microcapsule,etc., or parenterally in the form of injectable preparation such as asterile solution and a suspension in water or with otherpharmaceutically acceptable liquid. These preparations can be produced,for example, by mixing the mutein of the present invention with aphysiologically acceptable carrier, a flavoring agent, an excipient, avehicle, an antiseptic agent, a stabilizer, a binder, etc. in a unitdosage form which is generally accepted and required in a practicalpharmaceutical preparation. The effective component in the preparationis adjusted to such a dose that an appropriate dose is obtained withinthe specified range.

Additives miscible with tablets, capsules, etc. include a binder such asgelatin, corn starch, tragacanth and gum arabic, an excipient such ascrystalline cellulose, a swelling agent such as corn starch, gelatin andalginic acid, a lubricant such as magnesium stearate, a sweetening agentsuch as sucrose, lactose and saccharin, and a flavoring agent such aspeppermint, akamono oil and cherry. When the unit dosage is in the formof capsules, liquid carriers such as oils and fats may further be usedtogether with the additives described above. A sterile composition forinjection may be formulated by conventional procedures used to makepharmaceutical compositions, e.g., by dissolving or suspending theactive ingredients in a vehicle such as water for injection with anaturally occurring vegetable oil such as sesame oil and coconut oil,etc. to prepare the pharmaceutical composition. Examples of an aqueousmedium for injection include physiological saline and an isotonicsolution containing glucose and other auxiliary agents (e.g.,D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used incombination with an appropriate dissolution aid such as an alcohol(e.g., ethanol or the like), a polyalcohol (e.g., propylene glycol andpolyethylene glycol), a nonionic surfactant (e.g., polysorbate 80™ andHCO-50), etc. Examples of the oily medium include sesame oil and soybeanoil, which may also be used in combination with a dissolution aid suchas benzyl benzoate and benzyl alcohol. A buffer (e.g., phosphate buffer,sodium acetate buffer, etc.), a soothing agent (e.g., benzalkoniumchloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serumalbumin, polyethylene glycol, etc.), a preservative (e.g., benzylalcohol, phenol, etc.), an antioxidant, etc. can also be formulated. Thethus prepared liquid for injection is normally filled in an appropriateampoule.

The vector as described above, into which the DNA of the presentinvention is incorporated, can also be formulated in the same was asdescribed above, and usually used parenterally.

Since the thus obtained pharmaceutical preparations are safe and lowtoxic, the preparations can be administered to a human or mammal (e.g.,rat, mouse, guinea pig, rabbit, sheep, swine, bovine, cat, dog, monkey,etc.).

The DNA encoding the mutein of the present invention, or the vectorcontaining the DNA can be used for gene therapy for pulmonaryhypertension (particularly, primary pulmonary hypertension).

The dose of the prophylactic/therapeutic agent of the present inventionvaries depending on disease to be targeted, subject to be administered,route for administration, etc. In case of administering the mutein, theDNA or the antibody of the present invention to an adult patient (60 kgbody weight) with primary pulmonary hypertension in a form of injectablepreparation, it is advantageous to administer the active ingredientintravenously in a daily dose of about 0.01 to 30 mg, preferably about0.1 to 20 mg, and more preferably about 0.1 to 10 mg. For other animalspecies, the corresponding dose as converted per 60 kg body weight canbe administered.

In the specification and drawings, abbreviations of bases and aminoacids are based on the abbreviations of the IUPAC-IUB Commission onBiochemical Nomenclature or the conventional abbreviations used in theart, examples of which are shown below. An amino acid that has anoptical isomer takes its L form unless otherwise indicated.

DNA deoxyribonucleic acid cDNA complementary deoxyribonucleic acid Aadenine T thymine G guanine C cytosine RNA ribonucleic acid mRNAmessenger ribonucleic acid dATP deoxyadenosine triphosphate dTTPdeoxythymidine triphosphate dGTP deoxyguanosine triphosphate dCTPdeoxycytidine triphosphate ATP adenosine triphosphate EDTAethylenediaminetetraacetic acid SDS sodium dodecyl sulfate Gly glycineAla alanine Val valine Leu leucine Ile isoleucine Ser serine Thrthreonine Cys cysteine Met methionine Glu glutamic acid Asp asparticacid Lys lysine Arg arginine His histidine Phe phenylalanine Tyrtyrosine Trp tryptophan Pro proline Asn asparagine Gln glutamine pGlupyroglutamic acid

The substituents, protective groups and reagents, which are frequentlyused throughout the specification, are shown by the followingabbreviations.

Me methyl Et ethyl Bu butyl Ph phenyl TC thiazolidine-4(R)-carboxamideTos p-toluenesulfonyl CHO formyl Bzl benzyl Cl₂Bl 2,6-dichlorobenzyl Bombenzyloxymethyl Z benzyloxycarbonyl Cl-Z 2-chlorobenzyloxycarbonyl Br-Z2-bromobenzyloxycarbonyl Boc t-butoxycarbonyl DNP dinitrophenol Trttrityl Bum t-butoxymethyl Fmoc N-9-fluorenylmethoxycarbonyl HOBt1-hydroxybenztriazole HOOBt3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB1-hydroxy-5-norbornene-2,3-dicarboximide DCCN,N′-dicyclohexylcarbodiimide

Each SEQ ID NO (sequence identification number) in the Sequence Listingof the specification indicates the following sequence, respectively.

[SEQ ID NO: 1]

This shows the amino acid sequence of MCP-1.

[SEQ ID NO: 2]

This shows the amino acid sequence of 7ND-MCP-1.

[SEQ ID NO: 3]

This shows the nucleic acid sequence of DNA encoding MCP-1.

[SEQ ID NO: 4]

This shows the nucleic acid sequence of DNA encoding 7ND-MCP-1.

The present invention is described more specificically with reference tothe following examples, being not intended to limit the scope of thepresent invention thereto.

EXAMPLE 1

Effect of an antagonistic mutein of MCP-1 on the monocrotaline(MCT)-induced pulmonary hypertensive rat model.

Method: Normal rats (commercially available male rats with normal bloodpressure (6-8 week old)) were divided into four groups. The first groupis an untreated control group, the second group is an MCT (60 mg/kg,s.c.) treated control group, the third group is an MCT-treated groupadministered with a single dose of 7ND-MCP-1 (administeredsimultaneously with MCT-treatment), and the fourth group is anMCT-treated group administered with two doses of 7ND-MCP-1 (administeredsimultaneously with MCT-treatment and two weeks after theMCT-treatment). Three days before the gene introduction, 500 μl ofmarcaine was injected into the bilateral quadriceps femoris muscles ofthe rats, and three days later, 60 mg/kg MCT was subcutaneously injectedinto the MCT (60 mg/kg, s.c.) treated control group rats. To the rats inthe group administered with a single dose of 7ND-MCP-1 were injectedwith 500 μg of 7ND-MCP-1 expressing plasmids in the several discretesites in the bilateral quadriceps femoris muscles simultaneously with 60mg/kg MCT. To the rats in the group administered with two doses of7ND-MCP-1 were injected with 500 μg of 7ND-MCP-1 expressing plasmids inthe several discrete sites in the bilateral quadriceps femoris musclessimultaneously with 60 mg/kg MCT, and 14 days later, the rats wereinjected with 500 μg of 7ND-MCP-1 expressing plasmids in the severaldiscrete sites in the bilateral quadriceps femoris muscles. Changes inthe morphology of the cardiac ventricles and lumen sizes wereinvestigated by echocardiography and pressure in the right cardiacventricles was determined 1, 2, 3 and 4 weeks after the MCT-treatment.Cardiac weight, thickness of the right cardiac ventricle walls and lumensize of the cardiac ventricles were measured for the hearts extirpatedin the fourth week, and morphological analysis of the lungs wasperformed.

The 7ND-MCP-1 expression plasmid was generated as follows: 7ND-MCP-1cDNA was obtained by PCR, a well-known technique in the art, using theMCP-1 cDNA as the template (FLAG sequence can be added to the 3′terminus of 7ND-MCP-1); and the 7ND-MCP-1 cDNA was inserted between theBamHI and NotI sites of the pCDNA3 expression plasmid such that the 5′terminus of the cDNA is located at the BamHI site and the 3′ terminus islocated at the NotI site.

The results are shown below.

1. Effect on Pulmonary Hypertension

While the pressure in the right cardiac ventricles was 26.2±4.7 mmHg inthe untreated control group, it was increased to 69.7±2.1 mmHg fourweeks after the MCT-treatment. However, it was decreased to 37.3±4.6mmHg and 45.9±9.8 mmHg after the administration of a single dose and twodoses of 7ND-MCP-1 (an antagonistic mutein of MCP-1), respectively, thusconfirming a significant blood pressure lowering effect.

2. Effect on the Cardiac Weight and Morphological Changes in the Heart

While right ventricular hypertrophy, particularly increase of thicknessof the right cardiac ventricle walls, and lumen enlargement wereobserved in the MCT-treated group, these changes were prevented byadministration of 7ND-MCP-1.

3. Effect on Blood Vessels and Lung

Although significant hypertrophy of the tunica media was observed in thearterioles of the right cardiac ventricles in the MCT-administeredgroup, this change was suppressed by administration of 7ND-MCP-1. Whileinfiltration of large number of monocytes and macrophages predominantlyin the alveolar space was also observed in the MCT-treated group, thesechanges were suppressed by administration of 7ND-MCP-1.

In view of the effect of 7ND-MCP-1 administration on monocrotaline(MCT)-induced pulmonary hypertension and remodeling of pulmonary arteryand cardiac ventricle walls developing in association with thehypertension in the rats, an antagonistic mutein of MCP-1 is thought tobe a potential prophylactic or therapeutic agent for pulmonaryhypertension (primary pulmonary hypertension, in particular).

INDUSTRIAL APPLICABILITY

An agent comprising the antagonistic mutein of MCP-1 of the presentinvention, the DNA molecule having a nucleotide sequence encoding theantagonistic mutein of MCP-1, or the neutralizing antibody against MCP-1is useful as a pharmaceutical agent for preventing or treating pulmonaryhypertension (primary pulmonary hypertension, in particular).

1. A method for treating pulmonary hypertension, comprisingintramuscular administration of an effective amount of an nucleic acidencoding for a protein having the amino acid sequence of SEQ ID NO.: 2,or of a vector comprising said nucleic acid, to a mammal exhibitingpulmonary hypertension, wherein expression of the nucleic acid resultsin reduced blood pressure in said mammal.
 2. The method of claim 1wherein said nucleic acid is DNA.
 3. The method of claim 1 wherein saidvector is a plasmid vector.
 4. The method of claim 1 wherein said vectoris a viral vector.